Antioxidant Effects of DPP-4 Inhibitors in Early Stages of Experimental Diabetic Retinopathy

Hyperglycemia-induced oxidative stress plays a key role in the impairment of the retinal neurovascular unit, an early event in the pathogenesis of DR. The aim of this study was to assess the antioxidant properties of topical administration (eye drops) of sitagliptin in the diabetic retina. For this purpose, db/db mice received sitagliptin or vehicle eye drops twice per day for two weeks. Age-matched db/+ mice were used as the control group. We evaluated retinal mRNA (RT-PCR) and protein levels (Western blotting and immunohistochemistry) of different components from both the antioxidant system (NRF2, CAT, GPX, GR, CuZnSOD, and MnSOD) and the prooxidant machinery (PKC and TXNIP). We also studied superoxide levels (dihydroethidium staining) and oxidative damage to DNA/RNA (8-hydroxyguanosine immunostaining) and proteins (nitrotyrosine immunostaining). Finally, NF-кB translocation and IL-1β production were assessed through Western blotting and/or immunohistochemistry. We found that sitagliptin protected against diabetes-induced oxidative stress by reducing superoxide, TXNIP, PKC, and DNA/RNA/protein oxidative damage, and it prevented the downregulation of NRF2 and antioxidant enzymes, with the exception of catalase. Sitagliptin also exerted anti-inflammatory effects, avoiding both NF-кB translocation and IL-1β production. Sitagliptin prevents the diabetes-induced imbalance between ROS production and antioxidant defenses that occurs in diabetic retinas.


Introduction
Oxidative stress is defined as the imbalance between the formation and accumulation of reactive oxygen species (ROS) and the capacity of a biological system to neutralize them [1]. ROS as superoxide anion (O 2 •− ), peroxyl radical (ROO • ), and reactive hydroxyl radical ( • OH) are molecular species with one or more unpaired electrons that are consequently unstable, highly reactive, and can lead to protein, lipid, and DNA damage at high concentrations [2]. These products are produced physiologically and are necessary for the proper functioning of multiple cellular processes. The maintenance of physiological and non-damaging levels of ROS is regulated by antioxidant defenses [3]. Overproduction of ROS or defects in the antioxidant machinery can result in oxidative stress, which is strongly implicated in the development of several diseases, such as diabetes and its complications [4].
Diabetic retinopathy (DR) is one of the most frequent complications of diabetes and the leading cause of preventable blindness among the work-aged population in high-income Twenty diabetic male db/db (BKS.Cg-Dock7m +/+ Leprdb/J) mice and 10 nondiabetic mice (db/+; (BKS.Cg-Dock7m + Leprdb/+) were acquired from Charles River Laboratories (Calco, Italy) at 7 weeks of age for the experiments. The mutated leptin receptor carried by db/db mice gave rise to an obesity-induced type 2 diabetes phenotype. Mice were bred and maintained in the animal facilities of the Vall d'Hebrón Research Institute (VHIR). With the aim of minimizing variability, mice were randomly distributed (block randomization) into groups of two mice per cage in Tecniplast GM-500 cages (36 cm × 19 cm × 13.5 cm) under standard laboratory conditions at 22 ± 2 • C, with relative humidity of 50-60% and a 12 h light/dark cycle. The cages were equipped with nesting material, absorbent bedding (BioFresh Performance Bedding 1/800 Pelleted Cellulose, Absorption Corp, Ferndale, WA, USA), ad libitum food (ENVIGO Global Diet Complete Feed for Rodents, Mucedola, Milan, Italy), and filtered water. Glycemia was measured weekly through tail-vein blood sampling and a blood glucose meter (71371-80, FreeStyle Optium Neo; Abbott, IL, USA).
All animal experiments were directed in agreement with the European Community (86/609/CEE) and the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) for the utilization of laboratory animals. The Animal Care and Use Committee of VHIR (CEEA 14/21) authorized the present study.

Topical Ocular Treatment
Db/db mice aged 10 weeks received a topical ocular administration of 10 mg/mL sitagliptin phosphate monohydrate (Y0001812, Merck KGaA, Darmstadt, Germany) or vehicle eye drops [phosphate buffered saline (PBS)] for 2 weeks twice per day. Eye drops were randomly administered with the aid of a micropipette (5 µL), onto the superior corneal surfaces of both eyes of diabetic mice. On day 15, 1 h before euthanasia, animals received an additional dose of each treatment. Db/+ mice matched by age were used as the control group.

Retinal Tissue Processing
On day 15, mice were intraperitoneally injected with 200 µL of anesthesia, composed of a mix of ketamine (1 mL) (GmbH, Hameln, Germany) and xylazine (0.3 mL) (Laboratorios Calier S.A., Barcelona, Spain). Once anesthetized, animals that were intended to be used for immunofluorescence experiments were transcardially perfused with 4% paraformaldehyde (sc-281692, Santa Cruz Biotechnology, Dallas, TX, USA), while others were euthanized through cervical dislocation. Ocular globes were rapidly enucleated and differently processed depending on their purpose. All eyes, with the exception of those used for the immunofluorescence experiment, were dissected and the retinas obtained. Six retinas from each experimental group were submerged in 140 µL of TRIzol reagent (15596018, Invitrogen, Carlsbad, CA, USA) and assigned in different tubes until RNA extraction. Another six retinas from each group were immediately distributed in empty and distinct tubes until protein extraction. All of them were stored at −80 • C. Finally, the retinas from four animals of each group were not obtained after enucleation, and the entire ocular globes were fixed again in 4% paraformaldehyde for 5 h before paraffin embedding.

RNA Isolation and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay
Retinas (stored at −80 • C in 140 µL of TRIzol) were treated with DNase (18068015, ThermoFisher Scientific, Waltham, MA, USA) to avoid genomic contamination and were purified on an RNeasy MinElute column (74106, Qiagen, Hilden, Germany). After supernatant removal, RNA sediment was obtained and resuspended in 30 µL of RNAse free water (AM9937, ThermoFisher Scientific, Waltham, MA, USA). A Nanodrop spectrophotometer and an Agilent 2100 Bioanalyzer and were used for both RNA quantification and integrity, respectively. Reverse transcription of cDNA was assessed using Oligo(dT)18 Primers (SO131, ThermoFisher Scientific, Waltham, MA, USA) and a High-Capacity cDNA Reverse Transcription Kit (4368814, ThermoFisher Scientific, Waltham, MA, USA) and with the help of a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). RT-PCR was performed using SYBR Green PCR Master Mix (04707516001 Roche Diagnostics, Mannheim, Germany), specific primers (Table 1), a LightCycler480 System (05015243001, Roche Diagnostics, Mannheim, Germany), and 384-well optical plates (04729749001, Roche Diagnostics, Mannheim, Germany). Relative quantifications were obtained using the LightCycler480 SW 1.5.1 software and displayed as fold change versus control mice. B2m and Actin were used as housekeeping genes.

Primers
Gene ID Nucleotide Sequence

Statistical Analysis
Graph bars are displayed as the mean value followed by the standard error of the mean (SEM). Means of the different experimental groups were compared using both Students t-test and one-way ANOVA, which was accompanied by Bonferroni multiple-comparison post hoc test. Differences were considered statistically significant when p < 0.05.

Body Weight and Systemic Blood Glucose Levels of db/db Mice Not Modified after Topical Ocular Administration of Sitagliptin
Db/db mice presented significantly higher blood glucose levels than nondiabetic mice ( Figure 1A), confirming the diabetic status of the animals. No differences in body weight or blood glucose levels were found between diabetic mice treated with sitagliptin or vehicle eye drops ( Figure 1A,B). This finding indicates that all the observed effects can only be attributed to the direct effect of the drug on the retina and not to a systemic metabolic improvement.

Statistical Analysis
Graph bars are displayed as the mean value followed by the standard error of the mean (SEM). Means of the different experimental groups were compared using both Students t-test and one-way ANOVA, which was accompanied by Bonferroni multiple-comparison post hoc test. Differences were considered statistically significant when p < 0.05.

Body Weight and Systemic Blood Glucose Levels of db/db Mice Not Modified after Topical Ocular Administration of Sitagliptin
Db/db mice presented significantly higher blood glucose levels than nondiabetic mice ( Figure 1A), confirming the diabetic status of the animals. No differences in body weight or blood glucose levels were found between diabetic mice treated with sitagliptin or vehicle eye drops ( Figure 1A,B). This finding indicates that all the observed effects can only be attributed to the direct effect of the drug on the retina and not to a systemic metabolic improvement.

Sitagliptin Eye Drops Prevented the Antioxidant Deficiencies of the Diabetic Retina
Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a redox-sensitive transcription factor that plays a major defensive role in the NVU by modulating the expression of an-

Sitagliptin Eye Drops Prevented the Antioxidant Deficiencies of the Diabetic Retina
Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a redox-sensitive transcription factor that plays a major defensive role in the NVU by modulating the expression of antioxidant enzymes, regulating microglial dynamics, and protecting neurons and astrocytes from toxins [15]. It binds to the antioxidant response elements (ARE), which are located in the promoter region of genes that encode many antioxidant enzymes, such as copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX), or glutathione reductase (GR) [16]. In db/db mice, mRNA and protein levels of NRF2 were downregulated and topical sitagliptin treatment prevented decreased protein levels in all retinal layers (Figure 2A-C). Regarding all the aforementioned antioxidant enzymes, their retinal mRNA and protein levels were also downregulated in db/db mice, while sitagliptin prevented these abnormalities with the exception of catalase, on which it exerted a neutral effect ( Figure 3A-E). Topical administration of sitagliptin prevented this downregulation in all the retinal layers (i.e., MnSOD) or just in GCL (i.e., CuZnSOD) ( Figure 3D,E).  [16]. In db/db mice, mRNA and protein levels of NRF2 were downregulated and topical sitagliptin treatment prevented decreased protein levels in all retinal layers ( Figure  2A-C). Regarding all the aforementioned antioxidant enzymes, their retinal mRNA and protein levels were also downregulated in db/db mice, while sitagliptin prevented these abnormalities with the exception of catalase, on which it exerted a neutral effect ( Figure  3A-E). Topical administration of sitagliptin prevented this downregulation in all the retinal layers (i.e., MnSOD) or just in GCL (i.e., CuZnSOD) ( Figure 3D,E).  NRF2 regulates the transcription of other genes whose physiological expression is altered by hyperglycemia and oxidative stress, such as the gene encoding thioredoxin interacting protein (TXNIP) [17,18]. TXNIP is a prooxidant and proapoptotic protein upregulated in DR, which acts by inhibiting the ROS scavenging and thiol-reducing capability of the antioxidant enzyme thioredoxin (TRX). TXNIP expression has been strongly linked to hyperglycemia in retinal cell cultures, and its sustained expression over time leads to oxidative stress, inflammation, and premature cell death [18]. In db/db mice treated with vehicle, the number of TXNIP-positive cells was significantly higher than in control mice, while the values obtained in sitagliptin-treated db/db mice were similar to controls ( Figure 4A,B). Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 17 NRF2 regulates the transcription of other genes whose physiological expression is altered by hyperglycemia and oxidative stress, such as the gene encoding thioredoxin interacting protein (TXNIP) [17,18]. TXNIP is a prooxidant and proapoptotic protein up-regulated in DR, which acts by inhibiting the ROS scavenging and thiol-reducing capability of the antioxidant enzyme thioredoxin (TRX). TXNIP expression has been strongly linked to hyperglycemia in retinal cell cultures, and its sustained expression over time leads to oxidative stress, inflammation, and premature cell death [18]. In db/db mice treated with vehicle, the number of TXNIP-positive cells was significantly higher than in control mice, while the values obtained in sitagliptin-treated db/db mice were similar to controls ( Figure 4A,B).

Topical Administration of Sitagliptin Reduced the Aberrant Levels of Superoxide and Their Consequent Oxidative Damage to Biological Macromolecules in Diabetic Retinas
Excessive production of superoxide radicals is one of the main contributors to hyperglycemia-related oxidative stress in the diabetic retina [19]. Dihydroethidium (5-ethyl-5,6-dihydro-6-phenyl-3,8-diaminophe-nanthridine, hydroethidine, DHE) is a hydrophobic and uncharged molecule that has the capacity of crossing extra and intracellular membranes, where it can be oxidized by superoxide, giving rise to two different fluorescent products: ethidium, which is formed by specific redox reactions, and 2-hydroxyethidium, which is a specific adduct of superoxide. Therefore, DHE staining can be used to assess superoxide detection [20]. We found that relative DHE immunofluorescence expression was higher in the nuclear layers of diabetic mice than in the same layers of nondiabetic mice, and that sitagliptin reduced the hyperglycemia-related overproduction of O 2•− ( Figure 5A).

Topical Administration of Sitagliptin Reduced the Aberrant Levels of Superoxide and Their Consequent Oxidative Damage to Biological Macromolecules in Diabetic Retinas
Excessive production of superoxide radicals is one of the main contributors to hyperglycemia-related oxidative stress in the diabetic retina [19]. Dihydroethidium (5-ethyl-5,6-dihydro-6-phenyl-3,8-diaminophe-nanthridine, hydroethidine, DHE) is a hydrophobic and uncharged molecule that has the capacity of crossing extra and intracellular membranes, where it can be oxidized by superoxide, giving rise to two different fluorescent products: ethidium, which is formed by specific redox reactions, and 2-hydroxyethidium, which is a specific adduct of superoxide. Therefore, DHE staining can be used to assess superoxide detection [20]. We found that relative DHE immunofluorescence expression was higher in the nuclear layers of diabetic mice than in the same layers of nondiabetic mice, and that sitagliptin reduced the hyperglycemia-related overproduction of O 2 •− ( Figure 5A).
DNA/RNA oxidative damage [22]. Nitrotyrosine is another excellent biomarker of oxidative stress, and is formed as a result of protein nitration of free tyrosine residues by reactive peroxynitrite molecules [23]. In our study, both indicators of oxidative damage were significantly higher in the nuclear layers of diabetic retinas treated with vehicle than in the same layers of nondiabetic retinas. We observed that sitagliptin significantly prevented these DR-related abnormalities ( Figure 5B,C).

High PKC Presence in Diabetic Retinas was Reduced by Topical Treatment with Sitagliptin
Hyperactivation of protein kinase C (PKC) isoforms is produced by oxidative stress, and, in turn, contributes to oxidative stress. The PKC family is composed of 12 isoforms, in which PKC-α, -β, -δ, and -ε activation play a key role in the development of DR [10]. In the present study, the relative immunofluorescence expression of PKC-β and the number of PKC-δ positive cells were significantly higher in db/db mice treated with vehicle than in control mice. Treatment with sitagliptin eye drops avoided this abnormal increase of both PKC isoforms ( Figure 6A,B). High levels of ROS and reactive nitrogen species (RNS) facilitate their binding to biological such macromolecules as DNA, proteins, and lipids. These interactions promote damage to cell components, thus resulting in biological dysfunctions [21]. One of the most used markers is 8-hydroxyguanosine (8-OHG), the predominant product of DNA/RNA oxidative damage [22]. Nitrotyrosine is another excellent biomarker of oxidative stress, and is formed as a result of protein nitration of free tyrosine residues by reactive peroxynitrite molecules [23]. In our study, both indicators of oxidative damage were significantly higher in the nuclear layers of diabetic retinas treated with vehicle than in the same layers of nondiabetic retinas. We observed that sitagliptin significantly prevented these DR-related abnormalities ( Figure 5B,C).

High PKC Presence in Diabetic Retinas was Reduced by Topical Treatment with Sitagliptin
Hyperactivation of protein kinase C (PKC) isoforms is produced by oxidative stress, and, in turn, contributes to oxidative stress. The PKC family is composed of 12 isoforms, in which PKC-α, -β, -δ, and -ε activation play a key role in the development of DR [10]. In the present study, the relative immunofluorescence expression of PKC-β and the number of PKC-δ positive cells were significantly higher in db/db mice treated with vehicle than in control mice. Treatment with sitagliptin eye drops avoided this abnormal increase of both PKC isoforms ( Figure 6A,B).

Sitagliptin Exhibited Anti-Inflammatory Properties When Administered Topically in Diabetic Retinas
PKC pathway activation not only increases ROS production but also promotes nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Inactive NF-κB is located in the cytosol of almost all cell types, forming a complex with the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα). Several mechanisms, including hyperglycemia or oxidized proteins, can activate the enzyme IκB kinase (IKK) which phosphorylates IκBα. As result of phosphorylation, IκBα is ubiquitinated, dissociated from the NF-κB complex, and finally degraded by the proteasome. This signaling pathway provokes the activation of NF-κB and its consequent translocation to the cell nucleus, where it regulates inflammatory responses (i.e., cytokine production) [24]. Regarding our study, topical ocular administration of sitagliptin in db/db mice reduced NF-κB translocation in comparison to vehicle-treated db/db mice, showing similar results to the nondiabetic condition ( Figure 7). Furthermore, we observed an increase in mRNA and protein levels of interleukin 1 beta (IL-1β) in vehicle-treated diabetic mice, which was attenuated by sitagliptin ( Figure 8A,B).

Sitagliptin Exhibited Anti-Inflammatory Properties When Administered Topically in Diabetic Retinas
PKC pathway activation not only increases ROS production but also promotes nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Inactive NF-κB is located in the cytosol of almost all cell types, forming a complex with the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα). Several mechanisms, including hyperglycemia or oxidized proteins, can activate the enzyme IκB kinase (IKK) which phosphorylates IκBα. As result of phosphorylation, IκBα is ubiquitinated, dissociated from the NF-κB complex, and finally degraded by the proteasome. This signaling pathway provokes the activation of NF-κB and its consequent translocation to the cell nucleus, where it regulates inflammatory responses (i.e., cytokine production) [24]. Regarding our study, topical ocular administration of sitagliptin in db/db mice reduced NF-κB translocation in comparison to vehicle-treated db/db mice, showing similar results to the nondiabetic condition ( Figure 7). Furthermore, we observed an increase in mRNA and protein levels of interleukin 1 beta (IL-1β) in vehicle-treated diabetic mice, which was attenuated by sitagliptin ( Figure 8A,B).

Discussion
In the present study, we demonstrated the antioxidant and the anti-inflammatory properties of sitagliptin, a DPP-4i, in an experimental model of DR. These findings support and reinforce previous evidence showing the beneficial effects of topical administration of sitagliptin in early stages of DR [12,13].
Hyperglycemia-induced oxidative stress is one of the greatest threats to the retinal NVU. The oxygen consumption rate of the retina is extremely high, and it has an abundance of polyunsaturated acids, which are very susceptible to lipid peroxidation [9]. A clear relationship between the diabetic milieu and both ROS overproduction and deficiencies in the antioxidant machinery has been reported in multiple studies [25]. The physiological sources of ROS, mainly the mitochondrial electron transport chain (ETC) and the nicotinamide adenine dinucleotide (NAD + /NADH) phosphate (NADPH) oxidase family of enzymes (NOX), are disrupted in DR, leading to an excessive generation of ROS [26]. By contrast, the activity of antioxidant enzymes, which are directly or indirectly responsible for ROS and RNS scavenging, and the transcriptional functionality of the antioxidant factor NRF2 are both diminished in DR [27]. NRF2 is a major regulator of redox homeostasis, with an important role as a negative regulator of inflammation, attenuating oxidative stress by scavenging reactive oxygen species (ROS) and preventing genomic instability due to DNA damage. In the present study, we provide evidence that in db/db mice there exists a significant increase in superoxide levels and oxidative damage to DNA/RNA and proteins and a notable downregulation of NRF2 and the antioxidant enzymes CAT, GPX, GR, CuZnSOD, and MnSOD in comparison with db/+ nondiabetic control mice.
The capacity of sitagliptin to activate the NRF2/ARE pathway and ameliorate the pathological outcome of diseases where oxidative stress plays a key role has already been evidenced. For example, in a β-amyloid-induced rat model of Alzheimer's disease, sitagliptin improved cognitive status by its antioxidant effects mediated by the activation of NRF2 [28]. Sitagliptin was also able to increase the activities of some antioxidant enzymes, such as SOD, independently of their glucose-lowering abilities [29]. In the present study, we found that topical (eye drops) administration of sitagliptin modulated positively mRNA and protein production by the murine retina of both antioxidant elements: the NRF2/ARE pathway and the enzymatic defenses GPX, GR, CuZnSOD, and MnSOD. In addition, we found that sitagliptin prevented the upregulation of TXNIP (a prooxidant and proapoptotic protein) induced by diabetes, which plays a relevant role in the pathogenesis of DR [18]. Remarkably, we further detected that the topical administration of sitagliptin also reduced the RNA/DNA oxidative damage induced by RNS and ROS in the retinas of db/db mice.
It should be emphasized that the absence of differences in blood glucose levels between diabetic mice treated with vehicle or with sitagliptin eye drops support the idea that sitagliptin has antioxidant properties that cannot be attributed to its glucose-lowering capacity. Although there is some information regarding the antioxidant effect of sitagliptin when administered systemically [30,31], it is impossible to know whether this effect is due to the lowering effect of blood glucose levels or a direct effect of sitagliptin. In addition, the second possibility is unlikely, because it has been reported that sitagliptin in unable to cross the blood-retina barrier. Taken together, to the best of our knowledge, this is the first evidence of direct antioxidant effects of sitagliptin in the diabetic retina after its topical ocular administration.
Little is known about how DPP-4i exert their antioxidant and intrinsic functions. Since the increase in GLP-1 availability is one of the main consequences of DPP-4i administration, it could be postulated that GLP-1, rather than DPP4i, exerts the antioxidant effect. In fact, in previous studies we demonstrated that topically administered GLP-1 also has the capability of reducing oxidative damage in the same experimental model of DR [32]. However, the antioxidant effects of DPP4i have also been observed in in vitro models without GLP-1 [33]. In addition, we have found that sitagliptin, but not GLP-1 eye drops, improved the deficiencies in GPX and GR protein expressions and prevented the downregulation of CuZnSOD levels in the GCL layer. These findings suggest that sitagliptin may have GLP-1-independent mechanisms of action. These GLP-1-independent beneficial properties together with GLP-1-mediated effects, the lower price, and higher stability in comparison with GLP-1, make sitagliptin eye drops an ideal candidate to be tested in clinical trials for treating early stages of DR.
One of the biggest obstacles in the study of retinal NVU impairment in the context of early stages of DR is to establish a chronological order between the different disrupters (i.e., inflammation, glial activation, oxidative stress, neurodegeneration, and vascular leakage). PKC hyperactivation is one of the mechanisms that has been postulated as a possible link between hyperglycemia and oxidative stress in DR [34]. It is not still clear whether PKC activation contributes more to oxidative stress than oxidative stress contributes to its enhancement [10]. Geraldes et al. [35] reported that hyperglycemia promotes through PKC-δ two different fundamental pathways: cumulative ROS production and NF-κB activation. Notably, we have found that sitagliptin eye drops prevented the diabetesinduced upregulation of PKC-β and PKC-δ.
Hyperglycemia-induced oxidative stress leads to other pathological events, such as inflammation [36]. In the present study, we observed that topical administration of sitagliptin reduced NF-κB translocation and the production of IL-1β, proving the antiinflammatory properties of DPP-4i. Likewise, Li et al. [37] demonstrated that linagliptin, another DPP-4i, reduced TNF-α accumulation and NF-κB activation in retinal endothelial cells. Gonçalves et al. [30] also proved sitagliptin's capability to reduce nitrosative stress and IL-1β in the retinas of Zucker Diabetic Fatty (ZDF) rats. However, in this case, since sitagliptin was administered by the oral route, the beneficial effects could be attributed to the improvement in blood glucose control.
As a limiting factor, it could be argued that the duration of treatment (2 weeks) was too short. However, all the papers testing the effectiveness of DPP-IV inhibitors and other molecules addressed to treat early stages of DR using topical administration have been performed using similar treatment duration [12,[38][39][40][41][42][43]. It should be noted that we are treating early stages of DR in which microvascular retinal lesions are still absent on fundoscopic examination. However, neurodegeneration and impairment of the neurovascular unit could already be present. These abnormalities can occur very shortly after the onset of diabetes, and the time needed for any treatment to prevent or arrest them in the clinical arena remains to be elucidated. Nevertheless, although we are assuming that the effect of sitagliptin would persist if the treatment were longer, specific studies addressed to confirm this assumption are needed.

Conclusions
Although a close relationship between the diabetic milieu and both ROS overproduction and deficiencies in the antioxidant machinery have been reported in multiple studies, the underlying mechanisms involved in diabetes-induced oxidative stress remain to be elucidated. Our results suggest that the db/db mouse model recapitulates well the imbalance between ROS production and antioxidant defenses that exists in the diabetic retina. In addition, we provide the first evidence of the effectiveness of topical (eye drops) administration of sitagliptin in preventing diabetes-induced oxidative stress in the retina. This finding, together our previous reports showing neurovascular protection of sitagliptin, suggest this therapeutic strategy is promising, and therefore specific clinical trials seem warranted.